Rotavirus (RV) belongs to the rotavirus genus belonging to the Reoviridae family, which is the main pathogen responsible for infant diarrhea and was found in duodenum from patients with gastroenteritis by Bishop in 1973 (Bishop, Davidson et al. 1973). Studies showed that more than 95% of children were infected with rotavirus at least once before 5 years old. According to statistics from WHO, up to 600,000 people died of rotavirus infection annually, cases of diarrhea reached up to 200 million; and in USA only, economic loss caused by rotavirus infection reached up to 100 million dollars annually (Hsu, Staat et al. 2005; Tate, Burton et al. 2011), resulting in serious financial burden and social burden.
Rotavirus is a nonenveloped RNA virus. The genome of rotavirus consists of 11 double-stranded RNA molecules which encode 6 structure proteins (VP1-VP4, VP6 and VP7) and 6 non-structure proteins (NSP1-NSP6) (Estes and Cohen 1989). Rotavirus is icosahedral, and its capsid consists of three concentric layers, i.e. the core layer consisting of VP1, VP2 and VP3, the inner capsid consisting of VP6, and the outer capsid consisting of VP4 and VP7. VP6 is a species-specific antigen, and depending on its antigenicity, rotavirus may be divided into 7 groups, i.e. rotavirus A-G, among which rotavirus A is the main pathogen responsible for diarrhea among infants and young children. The protein has a strong immunogenicity, and although it is not a neutralizing antigen, it can have a good immune protection (Sabara, Frenchick et al. 1994). VP4 and VP7 are the main neutralizing antigens, and, rotavirus A can be divided into serotype P and serotype G depending on the antigenicity of them, and can be divided into different genotypes depending on their genes. G type and P type are independent of each other and are also interacted; the common combinations include G1P[8], G2P[4], G3P[8] and G4P[8]; in recent years, G9P[8] and G9P[6] are more and more popular (Li, Liu et al. 2009).
There are not specific drugs for rotavirus yet, and safe and effective vaccines are the important means for control of rotavirus infection. After years of research undergoing three phases, i.e. monovalent attenuated vaccines, polyvalent gene recombinant vaccines, and genetically engineering vaccines, there are four rotavirus vaccines appeared in the market one by one, including tetravalent human-ape gene recombinant vaccine from Wyeth, monovalent attenuated vaccine from Lanzhou Institute, pentavalent human-bovine gene recombinant vaccine from Merck, and monovalent attenuated vaccine from GSK. However, these vaccines are attenuated live vaccines which have large potential safety hazard, and the vaccines from Wyeth were recalled due to intestinal intussusception half a year after being in the market (Murphy, Gargiullo et al. 2001); although the vaccines from MERCK and GSK were demonstrated to be safe and effective by a large number of clinical tests (Bernstein, Sack et al. 1999; Vesikari, Matson et al. 2006; Linhares, Velazquez et al. 2008; Vesikari, Itzler et al. 2009; Snelling, Andrews et al. 2011), in countries and regions with a high rotavirus mortality such as Asia and Africa, the protection efficiency was much lower than that in developed countries such as Europe and America (Armah, Sow et al. 2010; Zaman, Anh et al. 2010; Madhi, Cunliffe et al. 2011). More and more evidence showed that upon vaccination with these two vaccines, shedding of virus occurred and horizontal transmission of virus might occur (Anderson 2008; Rivera, Pena et al. 2011; Yen, Jakob et al. 2011). It was also shown in some studies that serious gastroenteritis might be developed in children with immunologic deficiency after vaccination with the vaccines (Steele, Cunliffe et al. 2009; Patel, Hertel et al. 2010). The vaccines from Lanzhou Institute have been commercially available for more than 10 years, and no serious problem is found yet; however, they can only prevent serious diarrhea, and cannot prevent rotavirus infection (Fu, Wang et al. 2007). Therefore, although delightful results are obtained in studies on attenuated vaccines for rotavirus, there are also some problems and the safety and effectiveness need to be further improved. It is imperative to develop more safe and effective vaccines. Non-replicating vaccines are the main direction for studies on rotavirus vaccines now, and genetically engineering vaccines attract much attention because of the characteristics such as low cost, safety and effectiveness.
Genetically engineering vaccines mainly refer to antigens of rotavirus expressed by genetically engineering methods, which are used to immunize animal or human so as to achieve immune protection. Such vaccines include nucleic acid vaccines, synthetic peptide vaccines, recombinantly expressed antigen vaccine, and virus-like particles (VLPs) vaccines. The effectiveness and safety of VLPs vaccines now have been sufficiently demonstrated in the case of HBV, HEV and HPV vaccines, and have become a new generation of candidate vaccines with the greatest research value, as well recognized globally. The studies on RV-VLPs vaccines started in 80s of the last century, and a lot of animal experimental results showed that RV-VLP vaccines had good protective effect and could mediate a broad heterogenic protection.
RV-VLP refers to virus-like particle consisting of structure proteins, which is similar to native virus particle in terms of shape and structure and retains the native conformation of virus particle without containing viral nucleic acids. The particles can be divided into two classes; one class is a trilayer particle consisting of VP2, VP4, VP6 and VP7, or a double-layered particle consisting of VP6 & VP4, VP7, both of which can stimulate the generation of neutralizing antibodies in organisms (Crawford, Estes et al. 1999; Jiang, Estes et al. 1999); and the other class is a double-layered particle consisting of VP2 and VP6, and a monolayer particle consisting of VP6, which cannot stimulate the generation of neutralizing antibodies in organisms as they contain no neutralizing antigen, but also have a good protective effect as they can stimulate enhanced cell immunity in organisms (Coste, Sirard et al. 2000; Yuan, Geyer et al. 2000; Nguyen, Iosef et al. 2003); since variation in VP6 is relatively low, the particle can lead to a broad heterogenic protection. Relative to the first class of particles, the second class of particles have the same protective effect, but comprise less components, which greatly reduces processing difficulty and cost and thus are more favored.
The key for VP2/6-VLP vaccine development is to produce homogeneous VLP samples efficiently in a large amount. Insect baculovirus expression systems are commonly used now, and the rotavirus structure proteins VP2 and VP6 co-expressed in the system may self-assemble into VLP (Bertolotti-Ciarlet, Ciarlet et al. 2003). However, eukaryotic expression systems have the shortcomings such as high cost, long period, complex operations, and low expression level, and non-specific proteins and nucleic acids are generally encapsulated during the assembly, and thus it is difficult to achieve high-efficient and controllable assembly (Palomares and Ramirez 2009). Although there are studies on in vitro assembly of rotavirus VLP particles, the further development of RV VLP vaccines are restricted due to low yield.
Prokaryotic expression system has advantages such as low cost and simple operation. However, since prokaryotic expression system lacks specific posttranslational modifications, many proteins form inclusion bodies in prokaryotic expression. In current, there are studies showing that structure proteins of rotavirus were expressed in prokaryotic system, including VP6, VP4 and VP7, which were either expressed in inclusion bodies and unable to be renaturated effectively (Zhao, Chen et al. 2011), or expressed in a fusion form (Choi, Basu et al. 2000). Although fusion expression is favorable for the purification of desired proteins, expensive enzymes are generally required for cleavage of fusion proteins. Thus, prokaryotic expression system is not suitable for large-scale production.
Therefore, this field still demands techniques with low cost which can achieve high-efficient and controllable assembly and produce rotavirus structure proteins and virus-like particles at a large scale.